The major objective of this application is to continue our investigation on the role of ammonia and volatile aliphatic amines, in the normal regulation of gastrin release and the contribution of these amino acid metabolites to conditions associated with hypergastrinemia and antral gastritis. A major effort will be made to purify gastrin secretory granules from the rat antral mucosa either by density gradient centrifugation or affinity chromatography. Once isolated we will characterize the gastrin molecular forms stored within the granules, and the presence of key enzymes and proteins that may play a role in gastrin signaling and release which include: Annexin IV, H+-ATPase, and amino acid decarboxylase(s). We will employ the pH-sensitive fluorescent dye, acridine orange together with H+-ionophores to investigate the pH gradient across the endocrine-secretory granules, and the importance of ammonia/amines, calcium, and other putative intracellular mediators in the dissipation of this pH gradient and gastrin discharge. We will employ both the purified secretory granule fraction and synthetic membranes to investigate the pH-dependence of the hydrophobic bonding of gastrin to membrane phospholipids. We also plan to investigate granule swelling and fusion in response to these Annexin family (as Annexin IV appears to be present in the G cell) in the mediation of calcium-induced granule fusion and gastrin release will also be explored. In parallel with these studies we will perform experiments on isolated rat antral G cells and cultured human gastrinoma cells together with pH- and calcium-sensitive fluorescent dyes to assess the effects of ammonia, amines and amino acids on cytosolic pH and calcium levels, and how these changes relate to gastrin release. Lastly, we will intensely investigate the role of the excessive chronic accumulation of amines and ammonia in the gastric juice of rats in the development of severe hypergastrinemia in a number of animal models of gastritis and ulcer disease including: l) Taenia taeniaformis infection; 2) acute and chronic renal insufficiency; 3) H. felis infection; and 4) luminal (dietary) ammonia loading. These experiments which will employ RIA, immunocytochemical and molecular biological techniques should provide insight into the importance of gastric ammonia and amine accumulation in the changes in gastrin homeostasis seen clinically as a result of renal insufficiency and H. pylori infection.